Methods &amp; preperations of the latex from the croton species

ABSTRACT

The invention herein describes a procedure for organically extracting the lipophilic components from plants of the Family Euphorbaciae, specifically but not limited to the genus Croton. The extraction (CGO 110), is deplete of the normal proanthocyanidin content found in the parent material, yet retains its pharmacocological abilities and unlike the parent material, is selectively cytotoxic to cancer cells. The depletion of the proanthocyanidin components makes the product more amenable to preparations for the benefit of ameliorating both human and animal disease.

[0001] This application claims benefit of U.S. Provisional Applicationserial number 60/416,751 filed on Oct. 5, 2002.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] Sangre de grado or Sangre de drago, also known as “Dragon'sBlood,” is a viscous latex sap derived from the bark of various Crotonspecies (C. dracanoides, C. erythrochilus, C. gossypifilius, C.lechleri, C. palanostigma, C. sakutaris and C. urucurana) indigenous tothe South American rainforests. This latex has a deep red or burgundycolor that is attributed to its substantial proanthocyanidin content,estimated to being approximately 90% of the solid constituents of thesap. Ethnomedically, the latex is topically applied for the treatment ofpain and itching associated with insect bites and stings, as well asplant reactions. It is applied to the gums of patients after toothextractions, is utilized as a vaginal wash in the case of excessivebleeding and in the treatment of herpes where it is applied topically.It is also applied to open wounds as an anti-infective and as acicatrizant to accelerate the healing process. This latter effect mayresult from its constitutive taspine and crolechleric acid. It is takeninternally for a variety of distressing gastrointestinal symptoms,including the treatment of diarrhea, ulcers, vomiting and gutinflammation, as well as throat infections, tuberculosis and rheumatism.Oral intake is also associated with the ethnomedical application forcancer.

[0004] These traditional applications within South America cultures areless likely to be used in the Western world because of severalconstraints. Primarily, Sangre de grado's intense color, as suggested bythe reference to blood in its name, limits its ability to be usedtopically. In addition, Sangre de grado discolors and stains clothing ina similar manner as red wine, another proanthocyanidin rich extract. Ameans of reducing the proanthocyanidin content (and hence color) of thelatex whilst retaining its useful biological properties would representa significant improvement over the traditional botanical and allow formore widespread application.

[0005] The proanthocyanidins have been implicated as the mediators ofSangre de grado's antidiarrheal properties through the prevention ofcAMP mediated epithelial secretion. However, recent evidence suggeststhat Sangre de grado attenuates these epithelial secretory mechanisms bypreventing the activation of sensory afferent nerves that promotediarrhea, local inflammation, edema, as well process signals to thebrain for pain, nausea and itching. Capsaicin, the active component ofchili peppers, stimulates these sensory afferent nerves and Sangre degrado has been shown to impair capsaicin-induced epithelial secretion ofelectrolytes.

SUMMARY OF THE INVENTION

[0006] Aspects of the invention are summarized below to aid in theunderstanding of embodiment(s) of the invention and the application.Yet, the invention is fully defined by the claims of the application.

[0007] The latex or sap derived from the bark of the Croton species ofthe South American rainforests is associated with various ethnomedicalapplications including the treatment of cancer, diarrhea,gastrointestinal distress, pain and itching. While effective for theseindications the traditional ethnomedicine has undesirable effects thatlimit its use.

[0008] The present invention generally comprises an extract compositionderived from the latex or sap of the Croton species that retainsdesirable medicinal benefits despite reduced proanthocyanidin content.The extract disclosed herein retains the ability to inhibit emesis andactivation of sensory afferent nerves. The extract furthermorediscriminately promotes cancer cell death, unlike the parent material,at concentrations that fail to promote cell death in normal cells. Theextract composition retains its desirable properties but has a reducedcytotoxicity signifying an improvement over the parent botanical.

[0009] The extract composition is further incorporated into abiologically active dosage unit forming a beneficial wound-healingcomposition.

BRIEF DESCRIPTION OF THE FIGURES

[0010]FIG. 1. The extraction process significantly (“*”) reduces theproanthocyanidin content of the parent latex (SdG). When combined in abase vehicle, such as Aloe barbadensis shown here, the extract (CGO 110)produced a mixture absent of the intense color seen in similarpreparations with the parent latex. This change, which is readilyquantifiable by spectrophotometer, negates the discolorizing (i.e.staining) properties commonly associated with proanthocyanidins and theparent latex and allows for practical dermatological preparations;

[0011]FIG. 2. The prototypical activator of sensory afferent nerves,capsaicin, was topically applied to the mucosal surface of the stomachin anesthetized rats and mucosal blood flow measured by a Laser DopplerFlow meter. The marked increase in mucosal blood flow induced by 300 μMcapsaicin was prevented by either the parent material, SdG, or itsorganic extract, CGO 110 deplete of proanthocyanidins at doses of 2 and0.2 mg/ml, respectively, indicating that the organic extract retains theability to effectively prevent the activation of sensory afferentnerves;

[0012]FIG. 3. Using a well-established ferret model of post-operativecomplications of nausea, emesis and itch induced by morphine, theorganic extract CGO 110 was administered intraperitoneally (3 mg/kg) toferrets 15 minutes prior to the subcutaneous injection of 0.05 mg/kg ofmorphine-6-glucuronide (M6G). Administration of M6G caused a significantnumber of vomiting (2.2±0.4) and retching (10±1.2) incidences in thecontrol group while in those animals pre-treated with CGO 110, thenumber of these episodes was virtually abolished (vomiting 0.6±0.3;retching 2.2±0.7, P<0.05). It is clear that this organic extractionprocedure contains active components and is effective in the treatmentof emesis and itch.

[0013]FIG. 4. The selective cytotoxic ability of CGO 110 was tested invitro in cancerous cells from the gastrointestinal tract (AGS: stomach)and also in both normal macrophages and normal intestinal epithelialcells (IEC-18). In normal cells, Sangre de grado caused significant celldeath in both macrophages and IEC-18 cells while the same concentrationsof the organic extract CGO 110 did not. In stomach cancer cells (AGS),both CGO 110 and Sangre de grado were cytotoxic and the extract was morepotent than the parent botanical. Treatment of stomach cancer cells(AGS) with both CGO 110 and Sangre de grado caused cytotoxicity (celldeath), and the lipidic extract, CGO 110, was more potent than theparent botanical [the “*” denotes a significant difference between theSangre de grado and organic extract CGO 110 formulations (P<0.05)].Collectively, these results indicate that CGO 110 is selectivelycytotoxic to cancerous cells compared to the parent botanical, therebyrepresenting a marked improvement in safety.

DESCRIPTION OF AN EMBODIMENT

[0014] Extraction Procedure

[0015] According to one aspect of this invention, a process that retainsand concentrates the lipophilic components while reducing thehydrophilic proanthocyanidin content of the plant material resolves afamily Euphorbaciae extraction. This extraction process significantlyreduces the extracted composition of the hydrophilic proanthocyanidins,and hence its intense burgundy color, making it more amenable to topicalhealth care preparations. Furthermore, the product of this lipidicextraction, CGO 110, is selectively cytotoxic to cancerous cells, unlikethe parent material, representing an improvement in safety andsuggesting applications in the treatment of cancerous cells. Preferredmethods to accomplish the aforementioned family Euphorbaciae extractionare described by the procedures below but it is contemplated that askilled practitioner could device obvious variations of the proceduresgiven the disclosure herein and the desired results.

[0016] Extraction Process 1.

[0017] Latex, or sap from Croton species is mixed with an organicsolvent. The preferred organic solvent is ethyl acetate although otherorganic solvents can be used as would be obvious to the ordinarilyskilled practitioner in light of the disclosure herein. In otherembodiments, the preferred organic solvent is isopropanol, achloroform/Methanol mixture, or an equivalent thereof. The organicsolvent is added to the latex in a 1:1 proportion. In the preferredextraction process the solvent latex combination is agitated.

[0018] The preferred agitation method is stirring although otheragitation methods are also contemplated to be effective. Followingagitation, the mixture is settled, or allowed to settle into distinctphases including at least an organic layer and an aqueous layer. Theorganic phase or layer is comprised largely of solute lipophilicmaterials, representing the active constituent, and a significantlyreduced quantity of proanthocyanidin components relative to thepre-agitation step. The organic layer is separated from the aqueouslayer for further processing pursuant to the preferred extractionprocess.

[0019] Moreover, it is common to find a gel-like substance in theorganic layer at the interface of the aqueous and organic layers. Thisgel substance is characterized as having a dark brown and purple colorand comprises hydrophilic constituents trapped with water. In thepreferred process the gel substance is processed further to separate anyactive lipophilic constituents from the hydrophilic constituents. Thepreferred manner of processing the gel substance is the addition of adrying agent to the organic layer or the gel substance. The preferreddrying agent is magnesium sulfate in a concentration of 0.5-5 g/L ofcontaminant gel. It is contemplated that other equivalent drying agentsat relative effective concentrations would also be effective and wouldbe obvious to the ordinarily skilled practitioner in light of thedisclosure herein and with undue experimentation.

[0020] The addition of the drying agent results in a precipitant, whichtraps water and hydrophilic constituents or water-based colored chemicalcontaminants. The precipitant can be readily separated from thehydrophilic constituents by filtration or other techniques known toseparate precipitants. Actual laboratory procedures achieved acceptableresults using a Whatman #4 filter paper or an equivalent.

[0021] The steps of organic extraction, mixing with a drying agent andfiltration may be repeated up to three times to accomplish a thoroughextraction of the active lipophilic constituents. At this point in theprocess, the lipophilic materials are solutes contained within theorganic solvent, which are concentrated by evaporation of the solvent byone of several procedures, such as vacuum drying, freeze drying orheating. Actual heating up to 60 degrees Celsius produced acceptabledrying results.

[0022] The organic layer composition thus processed is rich inlipophilic materials but largely clear of hydrophilic contaminants.Following the extraction process, the color of the organic layer can becharacterized as a rose. Moreover, the reduced proanthocyanidin contentis quantifiable spectrophotometrically. Relative absorbance of theextraction in the visible spectrum was compared to the absorbency peakof the parent latex (414 nm) in the visible range.

[0023] At a concentration of 1 mg of extracted latex to 1 mL of waterthe disclosed process yielding the extraction (CGO 110) results in a 4.3fold reduction in absorbance at 414 nm, as indicated in FIG. 1. Thisassessment was repeated 9 times with similar results achieved(significance difference P<0.0001, as denoted by the “*”). Similarlywhen sangre de grado or the extraction (CGO 110) at a concentrations of200 μg per mL of aloe vera gel were applied to aloe vera gel to mimictheir administration as topical products, there was also a significantlylower color response with the extracted sangre de grado, CGO 110 vs. theparent botanical (*P<0.0001). See FIG. 1. Estimates from the absorbencymeasurements indicate that the proanthocyanidin content was reduced byat least 90% relative to the nonextracted parent latex.

[0024] Extraction Process 2.

[0025] The latex from the Croton species is dried to its residual solidmatter by methods such as heating, air-drying, vacuum or freeze-drying.The dried latex is rich in proanthocyanidin compounds and thereforecharacterized by a dark burgundy color. To the dried latex matter theorganic solvent, ethyl acetate or an equivalent, is added. The driedlatex and organic solvent mixture is agitated and the organic solvent isremoved for further processing according to the procedure described inExample 1. This process may be repeated up to three times to accomplisha thorough extraction all lipophilic materials in the organic layer andsolvent. If any water bearing contaminants are present, the addition ofdrying agent followed by filtration as noted above, will remove thesecontaminants. Removing the ethyl acetate through various methodsincluding heating, air-drying, vacuum or freeze-drying then isolates thesolutes contained within this organic extract.

[0026] The extraction thus processed according to the disclosedprocesses is characterized by a significant reduction ofproanthocyanidin compounds. The reduction of the proanthocyanidincompounds leaves the extraction significantly diminished in colorproducing compounds and yet amenable to health care applications.

[0027] Reduced Proanthocyanidin Content and Color Reactions

[0028]FIG. 1 illustrates the extent of proanthocyanidin depletionaccomplished by the extraction processes described herein. Relativeabsorbency of family Euphorbaciae latex Sangre de Grado (SdG) iscompared against a similar quantity of the latex that has been processedaccording to one of the procedures disclosed herein (CGO 110). As shownin FIG. 1, the extraction processes significantly diminishes theproanthocyanidin compounds or content compared to the parent latexmaterial and confirmed by a significant (500%) reduction in absorbencyin the 390 to 430 nm range. Since this wavelength range is within thehuman visible range, the extraction representing a significant reductionin visible color of this organic extract compared to the parentmaterial.

[0029] The presence of the proanthocyanidins in the parent latexprovides a rich burgundy color to the ethnomedicine, however it alsoresults in the generation of an intense “chocolate” color when combinedwith various base vehicles, including Aloe barbadensis (aloe vera)gel—and can thus act to stain various materials and textiles. Incontrast, the mixture of the organic extract (CGO 110) with a similarbase vehicle significantly reduces this color reaction, which can bereadily quantified spectrophotometrically. FIG. 1 illustrates thisresult and compares, a similar quantity of aloe barbadensis gel, whichhas insignificant absorbency in the 390 nm to 430 nm range, mixed with aquantity of the parent latex (SdG Gel), and mixed with a similarquantity of parent latex extracted by a process disclosed herein (CGO110 Gel).

[0030] Sangre de Grado has potential benefits as a topical applicant forvarious inflamed, itchy and irritated dermatological conditions.However, its inherent color due to a high proanthocyanidin content andthus the generation of an intense coloring when combined with basevehicles hinders its use for these applications. As the proanthocyanidincontent and thus coloring are significantly reduced by the disclosedprocesses, alone or in combination with other topical cremes, gels orbase vehicles, the extraction (CGO 110) signifies a marked improvementin the natural product and its uses.

[0031] Effects of the Organic Extract on Sensory Afferents

[0032] A prototypical activator of sensory afferent nerves, the nervesthat mediate the sensations of pain, itch, cough and nausea iscapsaicin, the pungent chemical found in chili peppers. Activation ofthese nerves by an activator such as capsaicin leads to a multitude ofresponses including vasodilation (mediated by the release ofneurotransmitters from these activated nerves that cause blood vesselsto relax), inflammatory cell recruitment, edema, and the sensations ofpain and itching. The Extraction (CGO 110) was tested to determine itsability to suppress sensory afferent nerve activation by testing itsability to inhibit capsaicin-induced increases in gastric blood flow.

[0033] The experiment involved the topical application of capsaicin tothe mucosal surface of the stomach in anesthetized rats and mucosalblood flow measured by a laser Doppler flow meter. As indicated in FIG.2, the marked increase in mucosal blood flow induced by 300 μM capsaicinwas prevented by either the parent material, SdG, or its organicextract, CGO 110 deplete of proanthocyanidins at doses of 2 and 0.2mg/ml, respectively. Thus, the organic extract described in thisapplication retains the ability to effectively prevent the activation ofsensory afferent nerves.

[0034] Effects on Morphine-Induced Emesis and Itch

[0035] Sangre de Grado has also been used ethnomedically for thetreatment of a variety of intestinal complications including diarrhea,ulcerations, cancer and emesis. Using a well-established ferret model ofpost-operative complications of nausea, emesis and itch induced bymorphine, the extraction (CGO 110) was administered intraperitoneally (3mg/kg) to ferrets 15 minutes prior to the administration ofmorphine-6-glucuronide (M6G), known to promote itching, retching andvomiting. The animals were monitored for sixty minutes. As shown in FIG.3, the subcutaneous injection of 0.05 mg/kg M6G caused a significantnumber of vomiting (2.2±0.4) and retching (10±1.2) incidences in thecontrol group. In those animals treated with extraction (CGO 110), thenumber of these episodes was virtually abolished (vomiting 0.6±0.3;retching 2.2±0.7, P<0.05). Itch as indicated by licking responses wasreduced from a control value of 16.9±2.3 episodes to 2.2±0.7 in CGO 110treated animals (P0.05). Given the utility of this model to predicttreatments for itch, nausea and vomiting, the extraction (CGO 110)contains active components and is effective in the treatment of emesisand itch.

[0036] Cytotoxicity: Cancer Cell Selectivity

[0037] While Sangre de grado has traditional uses in the treatment ofcancer, its utility is limited because it is equally toxic to bothnormal and cancerous cells. A process that could retain the ability ofSangre de grado to kill cancer cells but prevented these toxic effectson normal cells would represent a significant improvement over thetraditional medicine and a benefit to the treatment of disease in bothhumans and animals.

[0038] To test the selective cytotoxic ability of extraction (CGO 110)in vitro, cancerous cells from the gastrointestinal tract (AGS: stomach)and both normal macrophages and normal intestinal epithelial cells(IEC-18) were utilized. Cancerous GI cells were chosen based on Sangrede grado's traditional application for gastrointestinal complications.Cell death was determined by the MTT assay[3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide], whichassesses cell number by virtue of its oxidative or respiratory activityand the generation of a dye detectable at a wavelength of 550 nm.

[0039] As shown in FIG. 4, in normal cells, Sangre de grado causedsignificant cell death in both macrophages and IEC-18 cells while thesame concentrations of the organic extract CGO 110 did not. From this wecan determine that the lipidic extract CGO 110 has improved safety overthe parent botanical. Treatment of stomach cancer cells (AGS) with bothCGO 110 and Sangre de grado caused cytotoxicity (cell death), and thelipidic extract, CGO 110, was more potent than the parent botanical [the“*” in FIG. 4 denotes a significant difference between the Sangre degrado and organic extract CGO 110 formulations (P<0.05)]. Collectively,these results indicate that CGO 110 is selectively cytotoxic tocancerous cells compared to the parent botanical, thereby representing amarked improvement in safety.

[0040] Although the invention has been described in detail withreference to one or more particular preferred embodiments, personspossessing ordinary skill in the art to which this invention pertainswill appreciate that various modifications and enhancements may be madewithout departing from the spirit and scope of the claims that follow.

What is claimed is:
 1. A method of extracting the lipophilic componentsfrom plants of the family Euphorbaciae, comprising: combining plantmaterial from the family Euphorbaciae with an organic solvent; agitatingthe combination; settling the combination into distinct phases toresolve a layer substantially comprised of hydrophilic constituents andan organic layer substantially comprised of lipophilic constituents; andevaporating the organic layer to resolve the lipophilic constituents. 2.The method of claim 1 wherein the plant material is either in viscouslatex or at least partially dried latex from the family Euphorbaciae. 3.The method of claim 1 wherein the plant is of the genus Croton.
 4. Themethod of claim 1 wherein the organic solvent is selected from the groupconsisting of ethyl acetate, isopropanol and chloroform/Methanolmixture.
 5. The method of claim 1 further comprising, adding a dryingagent to the settled organic layer, prior to the step of evaporating theorganic layer, to further precipitate any remaining hydrophilicconstituents, and filtering the organic layer to resolve the lipophilicconstituents.
 6. The method in claim 5 wherein the drying agent selectedfrom the group consisting of, magnesium sulfate and sodium sulfate. 7.The method in claim 6 wherein the drying agent is magnesium sulfate andthe amount added per liter of organic layer is between about fivehundred milligrams (500 mg) to five grams (5 g) per liter.
 8. The methodin claim 5 wherein, after the step of filtering the organic layer, theorganic layer at a concentration of one milligram per milliliter (1mg/mL) of 50% (v/v) ethanol/water has an absorbance of about 0.120 AbsUnits in the wavelength range between about 390 nm and about 430 nm,relative to an absorbency of about 515 Abs Units within the samewavelength range.
 9. The method of claim 5 wherein the proanthocyanidincomponents are reduced by at least about 90% relative to the parentlatex.
 10. The method of claim 1 wherein the step of evaporating theprecipitate to resolve the hydrophilic constituents is selected from thegroup of evaporating method consisting of evaporation, spray drying,freeze drying, or vacuum drying.
 11. An extract of plant material fromfamily Euphorbaciae at a concentration of 1 mg/mL of 50% (v/v)ethanol/water having reduced relative UV absorbency between the range of390 nm and 430 nm.
 12. The extract in claim 11 wherein the relative UVabsorbency between the range of 390 nm and 430 nm is reduced by a factorof about at least 4.3 relative to the absorbency of the unextractedplant material from family Euphorbaciae
 13. The extract in claim 12wherein the UV absorbency between the range of 390 nm and 430 nm isabout 0.110 Abs Units relative to about 0.515 Abs Units for theunextracted plant material.
 14. An extract of plant material from familyEuphorbaciae at a concentration of about 1 mg/mL of carrier and havingreduced UV absorbency in the range of 390 nm to 430 nm relative to thesame concentration of unextracted plant material in the same carrier.15. The extract in claim 14 wherein the carrier is aloe barbadensis. 16.The extract in claim 14 wherein the UV absorbency between the range of390 nm and 430 nm is about 0.010 Abs Units relative to about 0.030 AbsUnits for the unextracted plant material.
 17. The extract in claim 11comprising, a pharmaceutical dosage unit composed of an extract offamily Euphorbaciae with reduced proanthocyanidin content and selectivecytotoxicity to cancerous cells.
 18. The extract in claim 11 comprising,a pharmaceutical dosage unit composed of an extract of familyEuphorbaciae that inhibits gastrointestinal distress manifested asemesis.
 19. The extract in claim 11 comprising, a pharmaceutical dosageunit composed of an extract of family Euphorbaciae that inhibits theactivation of sensory afferent nerves.